Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic profiles of purified microglia

Modern molecular neuroscience studies require analysis of specific cellular populations derived from brain tissue samples to disambiguate cell-type specific events. This is particularly true in the analysis of minority glial populations in the brain, such as microglia, which may be obscured in whole tissue analyses. Microglia have central functions in development, aging, and neurodegeneration and are a current focus of neuroscience research. A long-standing concern for glial biologists using in vivo models is whether cell isolation from CNS tissue could introduce ex vivo artifacts in microglia, which respond quickly to changes in the environment. Mouse microglia were purified by magnetic-activated cell sorting (MACS), as well as cytometer- and cartridge-based fluorescence-activated cell sorting (FACS) approaches to compare and contrast performance. The Cx3cr1-NuTRAP mouse model was used here to provide an endogenous fluorescent microglial marker and a microglial-specific translatome profile as a baseline comparison lacking cell isolation artifacts. All methods performed similarly for microglial purity with main differences being in cell yield and time of isolation. Ex vivo activation signatures occurred principally during the initial tissue dissociation and cell preparation and not the microglial cell sorting. Utilizing transcriptional and translational inhibitors during the cell preparation prevented the activational phenotype. These data demonstrate that a variety of microglial isolation approaches can be used, depending on experimental needs, and that inhibitor cocktails are effective at reducing cell preparation artifacts.

Spontaneous apoptosis of cells in therapeutic stem cell preparation exert immunomodulatory effects through release of phosphatidylserine

Mesenchymal stem cell (MSC)-mediated immunomodulation has been harnessed for the treatment of human diseases, but its underlying mechanism has not been fully understood. Dead cells, including apoptotic cells have immunomodulatory properties. It has been repeatedly reported that the proportion of nonviable MSCs in a MSC therapeutic preparation varied from 5~50% in the ongoing clinical trials. It is conceivable that the nonviable cells in a MSC therapeutic preparation may play a role in the therapeutic effects of MSCs. We found that the MSC therapeutic preparation in the present study had about 5% dead MSCs (DMSCs), characterized by apoptotic cells. Namely, 1 × 106 MSCs in the preparation contained about 5 × 104 DMSCs. We found that the treatment with even 5 × 104 DMSCs alone had the equal therapeutic effects as with 1 × 106 MSCs. This protective effect of the dead MSCs alone was confirmed in four mouse models, including concanavalin A (ConA)- and carbon tetrachloride (CCl4)-induced acute liver injury, LPS-induced lung injury and spinal cord injury. We also found that the infused MSCs died by apoptosis in vivo. Furthermore, the therapeutic effect was attributed to the elevated level of phosphatidylserine (PS) upon the injection of MSCs or DMSCs. The direct administration of PS liposomes (PSLs) mimic apoptotic cell fragments also exerted the protective effects as MSCs and DMSCs. The Mer tyrosine kinase (MerTK) deficiency or the knockout of chemokine receptor C–C motif chemokine receptor 2 (CCR2) reversed these protective effects of MSCs or DMSCs. These results revealed that DMSCs alone in the therapeutic stem cell preparation or the apoptotic cells induced in vivo may exert the same immunomodulatory property as the “living MSCs preparation” through releasing PS, which was further recognized by MerTK and participated in modulating immune cells.

Addition of Popular Exogenous Antioxidant Agent, PBN, to Culture Media May Be an Important Step to Optimization of Myogenic Stem/Progenitor Cell Preparation Protocol

The aim of the study was to modify human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) and demonstrate the optimal cell preparation protocol for application in post-infarction hearts. We used conditioned SkMDS/PC culture medium with α-phenyl-N-tert-butyl nitrone (PBN). SkMDS/PCs were cultured under hypoxic conditions and the results were compared to the standard ones. We observed a significant increase of CD-56 positive phenotypic marker the ability to form functional myotubes, increase in the proportion of young cells in cell primary suspensions, and a decrease in the percentage of apoptotic cells among PBN-conditioned cells in normoxia an hypoxia. We also observed significantly higher levels of SOD3 expression; maintained expression of SOD1, SOD2, and CAT; a higher level of BCL2 gene expression; and a rather significant decrease in Hsp70 gene expression in PBN-conditioned SkMDS/PCs compared to the WT population under hypoxic conditions. In addition, significant increase of myogenic genes expression was observed after PBN addition to culture medium, compared to WT population under hypoxia. Interestingly, PBN addition significantly increased the lengths of telomeres under hypoxia. Based on the data obtained, we can postulate that PBN conditioning of human SkMDS/PCs could be a promising step in improving myogenic cell preparation protocol for pro-regenerative treatment of post-infarction hearts.

IL-21 Optimizes the CAR-T Cell Preparation Through Improving Lentivirus Mediated Transfection Efficiency of T Cells and Enhancing CAR-T Cell Cytotoxic Activities

Adoptive immunotherapy using CAR-T cells is a promising curative treatment strategy for hematological malignancies. Current manufacture of clinical-grade CAR-T cells based on lentiviral/retrovirus transfection of T cells followed by anti-CD3/CD28 activation supplemented with IL-2 has been associated with low transfection efficiency and usually based on the use of terminally differentiated effector T cells. Thus, improving the quality and the quantity of CAR-T cells are essential for optimizing the CAR-T cell preparation. In our study, we focus on the role of IL-21 in the γc cytokine conditions for CAR-T cell preparation. We found for the first time that the addition of IL-21 in the CAR-T preparation improved T cell transfection efficiency through the reduction of IFN-γ expression 24–48 h after T cell activation. We also confirmed that IL-21 enhanced the enrichment and expansion of less differentiated CAR-T cells. Finally, we validated that IL-21 improved the CAR-T cell cytotoxicity, which was related to increased secretion of effector cytokines. Together, these findings can be used to optimize the CAR-T cell preparation.

A prospective phase I/IIa trial to evaluate the safety and efficacy of GTA002, an off-the-shelf, ex vivo-cultured allogeneic NK cell preparation in patients with acute myeloid leukemia in complete morphological remission who have measurable residual disease.

TPS7053 Background: Acute myeloid leukemia (AML) is a malignant disease with poor long-term prognosis in patients who cannot achieve morphological and molecular remission. Although insights into AML biology and treatment modalities have improved over recent years and even though many patients achieve morphological complete remission (CR), most are still relapsing. These relapses are due to residual leukemia stem cells that can be identified as minimal/measurable residual disease (MRD), with MRD serving as a predictive factor for relapse and mortality. Elimination of MRD in patients having reached CR is seen as essential for optimal and persistent clinical responses. A promising approach is the development of adoptive immunotherapies aimed at directly eradicating tumor cells using T-cells or natural killer (NK) cells. NK cells are part of the body’s innate immune system and play a key role in controlling viral infections and conducting tumor immunosurveillance. Furthermore, NK cells can be applied clinically in an allogeneic setting, enabling the supply of high numbers of immune effector cells, which were not exposed to cytotoxic chemotherapeutics. A proprietary ex vivo expansion and differentiation method in a fully closed, automated manufacturing platform was developed to generate GTA002, an “off-the-shelf” (allogeneic), cryopreserved NK cell preparation, generated from CD34+ hematopoietic stem and progenitor cells derived from umbilical cord blood. The safety and tolerability of the product was already demonstrated in a Phase I trial in elderly patients with AML (PMLA25) (Dolstra et al. 2017). Methods: We are currently conducting a prospective 2-stage, open-label, single arm, multicenter Phase I/IIa trial to evaluate the safety and efficacy of GTA002 in 33 adults with AML who are in CR with MRD and who are not proceeding to allogeneic HSCT (ClinicalTrials.gov Identifier: NCT04632316). Patients enrolled in the clinical trial receive a lymphodepleting conditioning regimen consisting of cyclophosphamide and fludarabine (Cy/Flu) followed by up to 3 NK cell infusions 4 days apart and will be followed up for 12 months. The dose escalation stage of the trial will assess the safety and tolerability of repeat NK cell infusions in a 3+3 design with 3 cohorts and a cumulative dose range of 325 to 3,000 x106 viable NK cells. The expansion stage will evaluate the safety, tolerability and efficacy of NK cell infusions in 24 additional subjects. The primary efficacy endpoint is the cumulative incidence of the MRD response and secondary efficacy endpoints include the duration of the MRD response, event-free survival, overall survival and cumulative incidence of relapse. Enrollment in the first cohort (one single NK cell infusion) started in December 2020. Clinical trial information: EudraCT number 2019-003686-17.

Study of the effect of Pioglitazone and Cetuximab on Cancer Stem-like Cellsenriched HT29 cell line

One of the major causes of cancer resistance to chemotherapy has been found to be the presence of Cancer Stem Cells (CSCs) in cancerous tissues. Probably, these cells are the source of cancer and the cause of malignancy and recurrence in the affected population. Therefore, it is possible to target CSCs to treat cancer. Since the percentage of CSCs in the total tumor mass is very low, so studies about these cells depend on their isolation and enrichment methods. Some studies have suggested that EMT induction in population of normal epithelial cells and cancer cells by inhibiting of E-cadherin, protects them against chemotherapy, anticancer and apoptosis drugs, moreover they get characteristics of CSCs. So in order to study CSCs can enrich them by inhibiting of E-cadherin in tumor population.In this study, we tried to examine how the effect of Pioglitazone and Cetuximab, two drugs used in chemotherapy of Colon Cancer, on CSCs enriched HT29 cell line (was called HT29-shE) in which CSCs were enriched with induction of EMT by inhibiting of E-cadherin using shRNA.For this purpose, after cell preparation EMT and CSCs markers in Pioglitazone and Cetuximab treated cells were assessed and compared with untreated cells using flow cytometry, real‐time PCR and microscopic monitoring. The findings showed mesenchymal morphology of HT29-shE changed to epithelial morphology after Pioglitazone and Cetuximab treatment, moreover E-cadherin expression increased and Vimentin expression decreased. In addition, expression of CSC markers (CD133+ and CD44+) were reduced in HT29-shE after treatment.

ATR-Spin: An open-source 3D printed device for direct cytocentrifugation onto Attenuated Total Reflectance crystals.

Infrared Spectroscopy (IR) enables the direct and rapid characterization of cells at the molecular level. Achieving a rapid and consistent cell preparation is critical for the development of Point-of-Care diagnostics...